a) Field of the Invention
The present invention relates to a method for modifying esterase enzymes with catalytic triads to vary K.sub.cat, K.sub.m and K.sub.cat /K.sub.m. It also relates to a method of varying the perhydrolysis/hydrolysis ratio of these enzymes especially for use in bleaching systems. It also relates to modification of the substrate specificity of esterase enzymes.
b) Background Information
A wide variety of esterase enzymes with catalytic site amino acids and in particular catalytic triads are well known. These include serine hydrolases and cystiene hydrolases. Broadly, these enzymes include esterases such as proteases, lipases, phospholipases, carboxyl esterases, wax esterases, cutinases and thio esterases.
These enzymes all act on esters participating in one of two reactions as follows: ##STR1##
These two types of reactions, perhydrolysis or hydrolysis are competing reactions and any given enzyme with the catalytic triad will produce both peracid and acid if the environment has both a water nucleophile source and a peroxygen nucleophile source. The ratio of these rates for any single enzyme is the perhydrolysis/hydrolysis ratio. These same enzymes also exhibit a given K.sub.cat, K.sub.m and K.sub.cat /K.sub.m. These are all measures of the kinetic properties of the enzyme and relate to such functions as substrate specificity and catalytic efficiency.
Substrate Specificity is a ubiquitous feature of biological macro molecules that is determined by chemical forces including hydrogen bonding, electrostatic, hydropholic and steric interactions for a given substrate and measured for a given substrate by Kcat/km and will range from substrate to substrate for a given enzyme. Jencks, W. P., in Catalysis in Chemistry and Enzymology (McGraw-Hill, 1969) pp. 321-436; Fersht, A., in Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp. 226-287. Substrate specificity study of enzymes has been limited to the traditional means of probing the relative importance of those binding forces. Although substrate analogs can be synthesized chemically, the production of modified enzyme analogs has been limited to chemically modified enzyme derivatives (Ann, Rev. Biochem, 54, 565-595 (1985) or naturally occurring mutants (Ann, Rev. Biochem, 46, 331-358).
Perhydrolysis is useful, among other things, in bleaching systems where the perhydrolysis reaction releases a peracid which is useful for fabric bleaching. European patent application 87-304963.9 describes such a method using an esterase and a peroxygen source to produce peracid for bleaching use. The enzymes described have catalytic triads, i.e. 3 catalytic site amino acids. The reaction is limited by the degree of the competing hydrolysis reaction with water and the enzyme to produce an acid.
Kcat/Km is a measure of the catalytic efficiency of an enzyme with a specific substrate. An enzyme will have a different substrate specificity for each substrate. It is desirable to have an enzyme with a greater (numerically large) K.sub.cat /K.sub.m for a given substrate to enable the use of the enzyme more efficiently on the target substrate.
Esterase Enzymes with catalytic triads and catalytic site amino acids are well known and available from a wide variety of sources such as from microbial, plant, insect or animal. These have a variety of useful properties as well as a variety of perhydrolysis/hydrolysis ratios and K.sub.cat, K.sub.m and K.sub.cat /K.sub.m values.
It would be useful if these enzymes could be modified to alter their perhydrolysis/hydrolysis ratio and K.sub.cat and K.sub.m so as to be more efficient in a desired reaction (e.g. altered substrate specificity). It would particularly be useful to convert carboxylic acid esters (triglycerides) into peracids by use of an enzyme wherein the competing acid producing reaction is minimized thus leading to a more efficient consumption of substrate.